Recently, InSphero Senior Application Scientist Sue Grepper wrote a blog on why we need better preclinical models for NASH drug discovery. I want to pick up where Sue left off to discuss in vitro methods and models that can improve our ability to predict the efﬁcacy and potential toxicity of drugs targeting steatosis, NASH, and fibrosis—and thus help reduce the attrition rate of clinical-stage therapies for these chronic liver diseases. Specifically, these models need to:
But first, let's briefly review why liver disease is a growing health concern today. Various stimuli, including diet and metabolic diseases, can alter hepatic lipid processing to induce intracellular accumulation of lipids in hepatocytes, a condition known as steatosis. If left unchecked, steatosis can progress to nonalcoholic steatohepatitis (NASH), which is characterized by hepatic necroinflammation and fibrosis. (Musso et al. 2016, Schuppan et al. 2018, Wong et al. 2018).
At present, no safe therapies have been approved for steatosis, NASH, or fibrosis. This is largely due to the lack of biologically relevant preclinical human models. The in vitro liver models most frequently used to study efficacy of anti-steatosis/NASH/fibrosis drugs are simple monolayer 2D cultures of primary hepatic stellate cells (HSC) or transformed HSCs (tHSC), such as LX-2. These monoculture models simply cannot recapitulate hepatocyte injury observed under fatty liver conditions (Santhekadur et al. 2018). Furthermore, HSCs and tHSCs are always activated in 2D culture environments, hindering assessment of the effects of relevant inducers of disease on the HSC phenotype. Culturing HSC in a 3D environment, however, has been shown to return these cells to their quiescent state (Leite et al. 2015).
IHC staining reveals the inherent decrease of HSC activation, shown by decreasing expression of α-SMA over 2 weeks in culture. Similarly, clearance of collagen deposition is captured in the decrease of collagen type 1 the same time period.
To better predict human drug efﬁcacy and toxicity, we need in vitro cell culture assays that exhibit the same molecular and functional ﬁngerprints seen in human diseases. We also need long-lasting cell-based models, suitable for longitudinal, repeat dose treatments that mimic chronic drug exposure in the clinic. We need an in vitro 3D system that contains the primary human liver cell types necessary to recapitulate key pathways of liver diseases prior to clinical trials (Santhekadur et al. 2018).
The liver team at InSphero has been developing liver disease models based on multicellular human liver microtissues containing hepatocytes (PHH), Kupffer cells (KC) and liver endothelial cells (LEC), and HSC for several years. We’ve engineered our 3D InSight™ Human Liver Disease Platform replicate steatosis, NASH and fibrosis. Thus far, we have successfully modeled phenotypes of diet-induced liver steatosis and fibrosis, and our NASH model is scheduled for release this fall. Potential applications for these liver disease models include drug efficacy and safety screening of anti-steatosis, anti-NASH and anti-fibrotic compounds.
InSphero scientists started with a human liver microtissue co-culture of PHH, KC, and LEC originally designed for detecting drug-induced toxicity. For liver disease modeling, we include additional cell types and/or clinically relevant agents to induce the desired liver disease. We also use an optimized liver media to support the long-term survival (~23 days) of all liver cell types within the microtissues. The extended lifespan of the microtissue in culture is necessary to model various liver diseases.
We developed a diet-induced steatosis model by using monoculture (PHH) and co-culture (PHH, KC and LEC), then loaded with free fatty acids (FFA) in specially formulated media containing normal or clinically relevant diabetic levels of sugars. This lipid loading results in formation of microvesicular and macrovesicular steatosis associated with NAFLD.
By adding HSCs, which are stimulated by by treatment with growth factor beta (TGF-β1) to this model, we established a liver fibrosis model. A well-known inducer of fibrosis, the TGF-β1 pathway activates SMAD2/3, which then translocates to the nucleus to induce the expression of pro-fibrotic genes, such as Col1A1, Col3A1, etc. Treatment of our 3D InSight™ Human Liver Fibrosis Model with TGF-β1 results in phenotypic changes similar to those observed in clinical liver fibrosis:
IHC staining demonstrates increased expression of pro-fibrotic markers, such as α-SMA, platelet derived growth factor receptor beta (PDGFRβ), and excessive deposition of collagens. Co-treatment with a TGFβR1 (ALK5) kinase inhibitor effectively blocks TGF-β1 -induced fibrosis and preserves the liver phenotype by blocking HSC activation.
IHC staining (brown, DAB) shows that that control liver disease microtissues exhibit presence of hepatocytes (albumin). TGF-β treatment induces elevated expression of α-SMA, Col I, and Col IV, accompanied by decreased hepatocyte function as detected by decreased albumin expression. Simultaneous treatment of TGF-β and ALK5 inhibitor halts the induction of fibrosis biomarkers and rescues hepatocyte function.
Our human liver fibrosis model facilitates testing of anti-fibrotic drugs that interfere with TGF-β-mediated fibrogenetic pathways. The ALK5-inhibitor assay enables researchers to evaluate effects of drug candidates on:
The 3D InSight™ Human Liver NASH Model of lipotoxic and inflammatory stress will build on what we’ve learned from our steatosis and fibrosis models. Cultured in a liver medium containing high sugar levels, this model will mimic key physiological aspects of NASH such as steatosis, inflammation and fibrosis. FFA and inflammatory stimuli in the 3D liver co-culture of PHH, KC, EC and HSC induce:
We characterized our NASH model on the morphological and functional level, by comparing it to established mechanisms of pathogenesis of NASH in the clinic.
The InSphero 3D InSight™ Liver Disease Platform has been specifically developed to provide pharma and biotech with the preclinical tools they need to screen and test candidate drugs before moving into clinical trials. Our data shows that InSphero disease models for liver steatosis, fibrosis, and NASH, effectively model the progressive stages of chronic human liver disease. These long-lived models enable researchers to conduct long-term, repeat dose studies that mimic how drugs will be employed in the clinic.
All 3D InSight™ Human Liver Disease Platform models are highly standardized and validated to ensure uniform size and cellular composition required for robust, reproducible results. Delivered in 96-well assay plates, designed for efficient handling and imaging, with 384 and organs-on-chips delivery formats scheduled to be released soon, these models are well-suited for for high-throughput efficacy drug screening, mechanistic studies of liver disease progression, discovery of novel therapeutics, as well as safety assessment of drugs in a disease-relevant background.
In my next blog, I'll delve into how NAFLD patients may be more susceptible to drug-induced liver injury (DILI), and how you can use our liver disease models for drug safety assessment. In the meantime, I encourage you to visit our website to learn more about 3D InSight™ Human Liver Disease Platform and our preclinical human models for diet-induced steatosis and fibrosis. We’ll post more details about our new NASH model soon.